Quantification of Neutrophil Extracellular Traps

Bovine PMN suspended in RPMI 1640 medium were confronted with B. besnoiti tachyzoites (4 h, 37°C, 5% CO₂) at a PMN/tachyzoite ratio of 1:6 (2×10⁵ PMN:1.2×10⁶ tachyzoites, 96-well format) in the presence of non-modified ATP (P1132, Promega, USA), non-hydrolyzable ATP (ATPγS; 0.05-50 µM; 4080, Tocris, UK), or purinergic receptor antagonists (see Table 1) at a concentration range of 0.1-100 µM. After incubation, sample supernatants were analyzed for “cell-free” NETs. The remaining cells at the well bottoms were estimated for “anchored” NETs according to (44 (link)). Therefore, picogreen (Invitrogen, Eugene, USA, 1:200 dilution in 10 mM Tris base buffered with 1 mM EDTA, 50 μl/well) was added to each supernatant or pellet sample. Extracellular DNA was quantified by picogreen-derived fluorescence intensities using an automated microplate reader (Varioskan, Thermo Scientific) at 484 nm excitation/520 nm emission as described elsewhere (26 (link), 30 (link)).